Susan V. Fuggle

THE DETAILED DISTRIBUTION OF MHC CLASS II ANTIGENS IN NORMAL HUMAN ORGANS

In a previous article we described the detailed tissue distribution of MHC class I antigens. In this study, we have used a monoclonal antibody, NFK1, to study the tissue distribution of MHC class II antigens. This antibody, which detects a monomorphic determinant common to the DR, SB, and DC molecules, was used to stain frozen sections of normal tissues from throughout the human body by a sensitive peroxidase-antiperoxidase immunohistological technique. Although previous studies, both in animal models and in human beings, have shown that class II antigens are expressed on a limited number of nonlymphoid tissues, our study has extended the spectrum of tissues on which this class of antigens is detectable. Epithelial cells in a number of organs were positively stained--these include the tongue, tonsils, epiglottis, trachea, small intestine, urethra, epididymis, and proximal renal tubules. Lymphatics throughout the body appeared to express class II antigens. Capillaries in brain, testis, and placenta appeared not to express class II antigens, but in the rest of the body they showed strong and uniform staining. These and other observations, and their implications, are discussed in relation to previously published studies.

The detailed distribution of HLA-A, B, C antigens in normal human organs.

We have used a monoclonal antibody, PA2.6, directed against the heavy chain of HLA-ABC antigens to study the detailed tissue distribution of MHC class I antigens. Normal tissues from throughout the human body were obtained fresh from organ donors or operative specimens and were snap-frozen in liquid nitrogen within 1-2 hr of removal. Frozen sections were then studied using a sensitive peroxidase-antiperoxidase immunohistological technique. The results of our study show that class I antigens could be detected on most, but not all, the nucleated cells in the body. They were only weakly detectable in several tissues including endocrine cells in the thyroid, parathyroid, pituitary and islets of Langerhans in the pancreas and on gastric mucosa, the myocardium, skeletal muscle, and hepatocytes in some of the specimens. Spermatozoa were positively stained in the testis, but as they moved up into the epididymis class I antigens were no longer detectable. We found that class I antigens were not detectable on corneal endothelium, some Brunners glands in the duodenum, villous trophoblast, central nervous system neurones, the exocrine portion of the pancreas, and acinar cells in the parotid. We conclude, therefore, that class I antigens are not ubiquitous, as previously thought.

Analysis of factors that affect outcome of primary cadaveric renal transplantation in the UK

BACKGROUND In the UK, kidneys are exchanged between centres on the basis of matching for HLA. We analysed various factors that might affect graft outcome to establish whether exchange of kidneys on this basis remains valid. METHODS 6363 primary cadaveric renal transplants carried out in 23 centres in the UK between 1986 and 1993 were used in the analysis. 6338 (99.6%) patients who underwent transplantation were followed up at 1 year. 5-year follow-up data were available for 2907 (97.8%) of the 2972 patients who survived to 5 years. We made random checks to validate the data. A multifactorial analysis with Coxs proportional hazards models was used to analyse factors that had a possible effect on graft outcome. To ensure that the analysis of matching was constant during the 8-year study, our analysis was based on the HLA antigens used for organ exchange (11 A locus antigens, 27 B locus antigens, and 12 DR locus antigens). We assessed overall outcome at 5 years and during three periods after transplantation at: 0-3 months, 3-36 months, and after 36 months. FINDINGS The following factors were significantly associated with graft outcome in the multifactorial analysis: year of graft, age of donor, age of recipient, whether the recipient had diabetes, cause of donors death, cold ischaemic time, transport of kidneys, transplant centre, and matching for HLA. The best outcome was achieved with kidneys that had no mismatches at HLA-A, HLA-B, and HLA-DR loci (000 mismatches). The next most favourable outcome was achieved with one mismatch at either A or B loci or one mismatch at both the A and B , but no mismatch at the DR locus (100, 010, or 110 mismatches). Age of the donor and recipient had a significant effect on transplant outcome: older age was associated with increased risk of graft failure. INTERPRETATION Various factors affect the outcome of primary cadaveric renal transplantation, particularly the age of the donor and the recipient. However, the effect of matching for HLA remains a strong one and fully justifies the continuing policy in the UK of exchanging kidneys on the basis of HLA matching, especially to recipients when there is a 000 mismatch for HLA between donor and recipient. On the basis of this analysis, a new allocation scheme for kidneys was introduced in the UK in 1998. During the first 9 months of the scheme, there has been a doubling of the number of HLA-000 mismatched kidneys transplanted.

Factors Affecting Graft and Patient Survival After Live Donor Kidney Transplantation in the UK

Background. The outcome after living donor renal transplantation is superior to that for deceased donor transplantation, but the results are not uniformly successful. The factors responsible for the variable outcome after living donor transplantation have not been well defined. Methods. UK Transplant Registry data were analyzed to determine the outcomes of 3142 first adult kidney transplants from living donors (71% genetically related and 29% unrelated) performed between 2000 and 2007 inclusive. Kaplan-Meier survival estimates were determined, and factors that might be associated with graft and patient survival were analyzed using Cox proportional hazards regression modeling. Results. Patient survival at 5 years was better for recipients of grafts from related than unrelated donors (97% vs. 93%, P=0.0002), but conversely graft survival was better in recipients of genetically unrelated grafts (93% vs. 89%, P=0.06). After adjustment for the factors found to influence graft and patient survival, these differences were no longer apparent. In contrast to the expectations, the degree of human leukocyte antigen-A, -B, and -DR mismatch did not influence graft survival. Increasing donor age (but not recipient age), recipient diabetes, and grafts from adult offspring were independently associated with poorer patient survival in the first 3 years after transplantation. Poorer graft survival was independently associated with donor age older than 59 years, and female recipients. Conclusions. Advanced donor age, but not human leukocyte antigen mismatch, is associated with poorer outcome after live donor kidney transplantation. However, the results of live donor transplantation remain superior to deceased donor kidney transplantation.BACKGROUND Mesenchymal stem cells (MSCs), also known as multipotent progenitor cells, release several factors that support cell survival and enhance wound healing. We hypothesized that MSC-secreted molecules would induce a trophic effect in pancreatic islet culture conditions. METHODS Pancreatic islets were co-cultured with MSCs, and ADP/ATP ratios, glucose stimulated insulin secretion (GSIS), and DNA fragmentation were evaluated to measure islet quality and viability in vitro. The induction of signal molecules related to the control of survival, function, and angiogenesis was also analyzed. Cell quality assays, DNA fragmentation assays, and islet transplantation into streptozotocin-induced diabetic mice were performed using MSC-conditioned medium (CM)-cultured islets. Furthermore, we identified soluble molecules within MSC-CM. RESULTS Islets co-cultured with MSCs demonstrated lower ADP/ATP ratios, and higher GSIS indexes and viability. Furthermore, co-cultured islets revealed higher levels of anti-apoptotic signal molecules (X-linked inhibitor of apoptosis protein, Bcl-xL, Bcl-2, and heat shock protein-32) and demonstrated increased vascular endothelial growth factor receptor 2 and Tie-2 mRNA expression and increased levels of phosphorylated Tie-2 and focal adhesion kinase protein. Islets cultured in MSC-CM demonstrated lower ADP/ATP ratios, less apoptosis, and a higher GSIS indexes. Diabetic mice that received islet transplants (200 islet equivalent) cultured in MSC-CM for 48 hr demonstrated significantly lower blood glucose levels and enhanced blood vessel formation. In addition, interleukin-6, interleukin-8, vascular endothelial growth factor-A, hepatocyte growth factor, and transforming growth factor-beta were detected at significant levels in MSC-CM. CONCLUSIONS These results suggest that the trophic factors secreted by human MSCs enhance islet survival and function after transplantation.

The role of mitochondria in ischemia/reperfusion injury

In organ transplantation, ischemia/reperfusion injury is a multifactorial process that leads to organ damage and primary graft dysfunction. Injury to the organ is mediated by a complex chain of events that involves depletion of energy substrates, alteration of ionic homeostasis, production of reactive oxygen species, and cell death by apoptosis and necrosis. There is increasing evidence that mitochondria play a role in this process because of the profound changes experienced during ischemia and reperfusion. Understanding the mechanisms that lead to mitochondrial damage may be important for developing strategies aimed at improving graft outcome. In this review, we examine the role of mitochondria in ischemia/reperfusion injury and the possible mechanisms that may contribute to organ dysfunction.

Variation in expression of endothelial adhesion molecules in pretransplant and transplanted kidneys--correlation with intragraft events.

Endothelial adhesion molecules are directly involved in the localization and migration of leukocytes from the circulation into tissues at sites of inflammation. We have compared the expression of PECAM-1 (CD31), ELAM-1, ICAM-1 (CD54), and VCAM-1 in pretransplant (n=20) and needle-core biopsies from renal transplants obtained during different clinical circumstances (n=42). PECAM-1 was consistently expressed on all endothelium in both pretransplant and transplant biopsies. In contrast, there was variation in endothelial expression of ELAM-1 and in proximal tubular expression of ICAM-1 and VCAM-1 between pretransplant biopsies. After transplantation induced expression of endothelial ELAM-1 and VCAM-1 and tubular induction of ICAM- 1 and VCAM-1 was detected. Induced adhesion molecule expression was frequently associated with focal leukocyte infiltration, and there was a significantly higher level of CD45 and CD25 positive cell infiltration in biopsies with induced adhesion molecule expression. The induction of adhesion molecule expression is evidence of endothelial activation in these transplant biopsies. Comparison of adhesion molecule expression and HLA-class II antigen expression revealed that induced tubular class II antigens may be detected in the absence of induced adhesion molecule expression.

Apoptosis in ischemia/reperfusion injury of human renal allografts.

BACKGROUND Ischemia/reperfusion injury of human renal allografts has a number of clinically significant consequences. A number of mechanisms of ischemia/ reperfusion injury have been elucidated, and there is evidence that apoptosis may be a contributing factor. METHODS To examine immediate posttransplant events, fixed tissue sections from paraffin-embedded wedge biopsy specimens taken before and after reperfusion of human renal allografts were stained using terminal deoxytransferase-mediated dUTP nick-end labeling to detect the DNA fragmentation characteristic of apoptosis. Thirty-six pairs of pre- and postreperfusion biopsy specimens were examined, 11 from living-related donor renal transplants and 25 from cadaveric donor transplants. RESULTS Quantitation of the terminal deoxytransferase-mediated dUTP nick-end labeling signal showed that significantly more apoptosis occurred in postreperfusion compared with prereperfusion biopsy specimens from cadaveric donor transplants, but a similar difference was not observed in living-related donor renal transplants. Furthermore, significantly more apoptosis was observed in postreperfusion biopsy specimens from cadaveric compared with living-related renal transplants. Postreperfusion biopsy specimens from kidneys that were cold preserved longer than 30 hr had a higher mean apoptosis score than those stored for less than 24 hr, but the result was not statistically significant. CONCLUSIONS Thus, apoptosis occurs predominantly as a result of reperfusion after cold preservation of cadaveric donor renal allografts and provides additional information regarding the extent of ischemia/ reperfusion injury in an organ. The clinical value of this information remains to be determined.

LOCALIZATION OF HLA-ABC AND DR ANTIGENS IN HUMAN KIDNEY

Monoclonal antibodies to human monomorphic class I and class II major histocompatibility complex (MHC) determinants have been used with immunofluorescence and immunoperoxidase techniques, to localize these antigens in normal human kidneys. HLA-DR antigen was located in the glomeruli (probably on endothelium as well as in the mesangium) and within the cells of cortical and medullary tubules. Dendritic cells in the renal inter-stitium stained brightly for the DR antigen and could be distinguished from the staining of capillary endothelium. The vascular endothelium of large vessels stained less densely for the HLA-DR antigen than for HLA-ABC antigens. The glomeruli stained intensely for the HLA-ABC antigens and diffuse staining of HLA-ABC antigens was also noted within renal tubular cells.

Cell adhesion molecules in clinical renal transplantation.

Leukocyte adhesion molecules are critically involved at a number of stages in immune and inflammatory responses, and their importance in the response to a renal allograft has been recognized for some years. They are involved in antigen presentation, in the cascade of events leading to extravasation of leukocytes into the allograft, in the subsequent migration of leukocytes through the extracellular matrix, and in the interactions between effector and target cells. Thus the adhesion molecules are highly attractive targets for therapeutic intervention in organ transplantation. Strategies have been explored to exploit the involvement of adhesion molecules in ischemia/reperfusion injury, allograft rejection, and the induction of immunological tolerance. Furthermore, the expression of a number of adhesion molecules is regulated by cytokines, and elevated levels may be detected both in transplant biopsies and as soluble forms measured in serum and urine. It has been proposed that these changes in levels might provide useful information in the diagnosis of allograft rejection and differentiation from other causes of graft dysfunction.

Tools for human leukocyte antigen antibody detection and their application to transplanting sensitized patients.

In recent years there have been major advances in the technology for the detection and definition of human leukocyte antigen antibodies. In this overview we describe the evolution in laboratory technology, the techniques currently available and consider their application in antibody specificity definition and in understanding a patients sensitization profile. We discuss the importance of antibody specificity definition in facilitating efficient national organ allocation and informing clinical discussion regarding the appropriate pathway for sensitized patients awaiting renal transplantation.